Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes.

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4+ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential.

Structures of Escherichia coli tryptophanase in holo and 'semi-holo' forms.

Two crystal forms of Escherichia coli tryptophanase (tryptophan indole-lyase, Trpase) were obtained under the same crystallization conditions. Both forms belonged to the same space group P43212 but had slightly different unit-cell parameters. The holo crystal form, with pyridoxal phosphate (PLP) bound to Lys270 of both polypeptide chains in the asymmetric unit, diffracted to 2.9 Šresolution. The second crystal form diffracted to 3.2 Šresolution. Of the two subunits in the asymmetric unit, one was found in the holo form, while the other appeared to be in the apo form in a wide-open conformation with two sulfate ions bound in the vicinity of the active site. The conformation of all holo subunits is the same in both crystal forms. The structures suggest that Trpase is flexible in the apo form. Its conformation partially closes upon binding of PLP. The closed conformation might correspond to the enzyme in its active state with both cofactor and substrate bound in a similar way as in tyrosine phenol-lyase.

Conformational changes and loose packing promote E. coli Tryptophanase cold lability.

BACKGROUND: Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One such enzyme is tryptophanase (Trpase) from Escherichia coli. Trpase is a pyridoxal phosphate (PLP)-dependent tetrameric enzyme with a Mw of 210 kD. PLP is covalently bound through an enamine bond to Lys270 at the active site. The incubation of holo E. coli Trpases at 2 degrees C for 20 h results in breaking this enamine bond and PLP release, as well as a reversible loss of activity and dissociation into dimers. This sequence of events is termed cold lability and its understanding bears relevance to protein stability and shelf life. RESULTS: We studied the reversible cold lability of E. coli Trpase and its Y74F, C298S and W330F mutants. In contrast to the holo E. coli Trpase all apo forms of Trpase dissociated into dimers already at 25 degrees C and even further upon cooling to 2 degrees C. The crystal structures of the two mutants, Y74F and C298S in their apo form were determined at 1.9A resolution. These apo mutants were found in an open conformation compared to the closed conformation found for P. vulgaris in its holo form. This conformational change is further supported by a high pressure study. CONCLUSION: We suggest that cold lability of E. coli Trpases is primarily affected by PLP release. The enhanced loss of activity of the three mutants is presumably due to the reduced size of the side chain of the amino acids. This prevents the tight assembly of the active tetramer, making it more susceptible to the cold driven changes in hydrophobic interactions which facilitate PLP release. The hydrophobic interactions along the non catalytic interface overshadow the effect of point mutations and may account for the differences in the dissociation of E. coli Trpase to dimers and P. vulgaris Trpase to monomers.

New tryptophanase inhibitors: towards prevention of bacterial biofilm formation.

Tryptophanase (tryptophan indole-lyase, Tnase, EC 4.1.99.1), a bacterial enzyme with no counterpart in eukaryotic cells, produces from L-tryptophan pyruvate, ammonia and indole. It was recently suggested that indole signaling plays an important role in the stable maintenance of multicopy plasmids. In addition, Tnase was shown to be capable of binding Rcd, a short RNA molecule involved in resolution of plasmid multimers. Binding of Rcd increases the affinity of Tnase for tryptophan, and it was proposed that indole is involved in bacteria multiplication and biofilm formation. Biofilm-associated bacteria may cause serious infections, and biofilm contamination of equipment and food, may result in expensive consequences. Thus, optimal and specific factors that interact with Tnase can be used as a tool to study the role of this multifunctional enzyme as well as antibacterial agents that may affect biofilm formation. Most known quasi-substrates inhibit Tnase at the mM range. In the present work, the mode of Tnase inhibition by the following compounds and the corresponding Ki values were: S-phenylbenzoquinone-L-tryptophan, uncompetitively, 101 microM; alpha-amino-2-(9,10-anthraquinone)-propanoic acid, noncompetitively, 174 microM; L-tryptophane-ethylester, competitively, 52 microM; N-acetyl-L-tryptophan, noncompetitively, 48 microM. S-phenylbenzoquinone-L-tryptophan and alpha-amino-2-(9,10-anthraquinone)-propanoic acid were newly synthesized.

Structural insights into cold inactivation of tryptophanase and cold adaptation of subtilisin S41.

A wide variety of enzymes can undergo a reversible loss of activity at low temperature, a process that is termed cold inactivation. This phenomenon is found in oligomeric enzymes such as tryptophanase (Trpase) and other pyridoxal phosphate dependent enzymes. On the other hand, cold-adapted, or psychrophilic enzymes, isolated from organisms able to thrive in permanently cold environments, have optimal activity at low temperature, which is associated with low thermal stability. Since cold inactivation may be considered "contradictory" to cold adaptation, we have looked into the amino acid sequences and the crystal structures of two families of enzymes, subtilisin and tryptophanase. Two cold adapted subtilisins, S41 and subtilisin-like protease from Vibrio, were compared to a mesophilic and a thermophilic subtilisins, as well as to four PLP-dependent enzymes in order to understand the specific surface residues, specific interactions, or any other molecular features that may be responsible for the differences in their tolerance to cold temperatures. The comparison between the psychrophilic and the mesophilic subtilisins revealed that the cold adapted subtilisins have a high content of acidic residues mainly found on their surface, making it charged. The analysis of the Trpases showed that they have a high content of hydrophobic residues on their surface. Thus, we suggest that the negatively charged residues on the surface of the subtilisins may be responsible for their cold adaptation, whereas the hydrophobic residues on the surface of monomeric Trpase molecules are responsible for the tetrameric assembly, and may account for their cold inactivation and dissociation.

The structure of apo tryptophanase from Escherichia coli reveals a wide-open conformation.

The crystal structure of apo tryptophanase from Escherichia coli (space group F222, unit-cell parameters a = 118.4, b = 120.1, c = 171.2 A) was determined at 1.9 A resolution using the molecular-replacement method and refined to an R factor of 20.3% (R(free) = 23.2%). The structure revealed a significant shift in the relative orientation of the domains compared with both the holo form of Proteus vulgaris tryptophanase and with another crystal structure of apo E. coli tryptophanase, reflecting the internal flexibility of the molecule. Domain shifts were previously observed in tryptophanase and in the closely related enzyme tyrosine phenol-lyase, with the holo form found in an open conformation and the apo form in either an open or a closed conformation. Here, a wide-open conformation of the apo form of tryptophanase is reported. A conformational change is also observed in loop 297-303. The structure contains a hydrated Mg(2+) at the cation-binding site and a Cl(-) ion at the subunit interface. The enzyme activity depends on the nature of the bound cation, with smaller ions serving as inhibitors. It is hypothesized that this effect arises from variations of the coordination geometry of the bound cation.

Membrane-catalyzed nucleotide exchange on DnaA. Effect of surface molecular crowding.

DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a central role in the timing of the primary initiations within the Escherichia coli cell cycle. A controlled, reversible conversion between the active ATP-DnaA and the inactive ADP forms modulates this activity. In a DNA-dependent manner, bound ATP is hydrolyzed to ADP. Acidic phospholipids with unsaturated fatty acids are capable of reactivating ADP-DnaA by promoting the release of the tightly bound ADP. The nucleotide dissociation kinetics, measured in the present study with the fluorescent derivative 3'-O-(N-methylantraniloyl)-5'-adenosine triphosphate, was dependent on the density of DnaA on the membrane in a cooperative manner: it increased 5-fold with decreased protein density. At all surface densities the nucleotide was completely released, presumably due to protein exchange on the membrane. Distinct temperature dependences and the effect of the crowding agent Ficoll suggest that two functional states of DnaA exist at high and low membrane occupancy, ascribed to local macromolecular crowding on the membrane surface. These novel phenomena are thought to play a major role in the mechanism regulating the initiation of chromosomal replication in bacteria.

Crystallization and preliminary X-ray analysis of the apo form of Escherichia coli tryptophanase.

Tryptophanase from Escherichia coli is a pyridoxal phosphate-dependent homotetrametic enzyme with a subunit weight of 52 kDa. It has been crystallized in the apo form by the hanging-drop vapour-diffusion method using polyethylene glycol 400 as a precipitant and magnesium chloride as an additive. The crystals belong to the orthorhombic space group F222, with unit-cell parameters a = 118.4, b = 120.1, c = 171.2 A. A 97.8% complete data set to 1.9 A resolution was collected at a rotating-anode source from a single frozen crystal. Packing-density considerations agree with a monomer in the asymmetric unit with a solvent content of 55%. Tryptophanase mutants W330F and Y74F were crystallized under the same conditions and the crystals diffracted to a resolution limit of 1.9 A. Data sets of wild-type crystals soaked with L-tryptophan or pyridoxal phosphate were collected, as well as of Y74F mutant soaked with both.

Bioadhesive grafted starch copolymers as platforms for peroral drug delivery: a study of theophylline release.

Nonirritant bioadhesive drug release systems based on starch-acrylic acid graft copolymers prepared by radiation of starch and acrylic acid mixtures with (60)Co were developed for buccal application. The release rate of theophylline (TPL), used as a model drug, depended on the ratio of starch to acrylic acid and on the presence of cations in the graft copolymers, but was practically not affected by the pH (between pH 3 and 7) of the dissolution medium nor by the type of starch used (corn, rice, or potato). Possible release mechanisms are discussed for specific conditions. In general, the release behavior of the graft copolymers was found to be non-Fickian, n value being between 0.6 and 0.96, suggesting that the release was controlled by a combination of tablet erosion and the diffusion of the drug from the swollen matrix. Incorporation of divalent cations into the graft copolymers led to a significant decrease in swelling erosion of the tablets as well as a substantial retardation of drug release. Highest work of adhesion was obtained with graft copolymers containing calcium ions as well as longer time of adhesion on dogs' gingiva.

Cold-induced enzyme inactivation: how does cooling lead to pyridoxal phosphate-aldimine bond cleavage in tryptophanase?

The phenomenon of cold scission or cold lability, which entails a widespread variety of oligomeric enzymes, is still enigmatic. The effect of cooling on the activity and the quaternary structure of the pyridoxal 5'-phosphate (PLP)-dependent enzyme, tryptophanase (Tnase), was studied utilizing single photon counting time-resolved spectrofluorometry. Upon cooling of holo-wild-type (wt) Tnase and its W330F mutant from 25 degrees C to 2 degrees C, a reduction in PLP fluorescence lifetime and rotational correlation time as well as inactivation and dissociation from tetramers to dimers were observed for both enzymes. Fluorescence anisotropy invariably decreased as a consequence of cooling, whether it was accompanied by a slight decrease in activity without significant dissociation, or by a substantial decrease in activity that was associated with either a partial or major dissociation. These results support the suggested conformational change that precedes the PLP-aldimine bond scission. It is proposed that cold inactivation is initiated by the weakening of hydrophobic interactions, leading to conformational changes which are the driving force for the aldimine bond cleavage.